1. Field of the Invention
The invention relates to novel DNA constructs, plant cells containing the constructs and plants derived therefrom.
In particular it relates to the modification of carotenoid metabolism in plants using DNA constructs.
2. Description of the Prior Art
The modification of plant gene expression has been achieved by several methods. The molecular biologist can choose from a range of known methods to decrease or increase gene expression or to alter the spatial or temporal expression of a particular gene. For example, the expression of either specific antisense RNA or partial (truncated) sense RNA has been utilized to reduce the expression of various target genes in plants (as reviewed by Bird and Ray, 1991, Biotechnology and Genetic Engineering, Reviews 9: 207-227). These techniques involve the incorporation into the genome of the plant of a synthetic gene designed to express either antisense or sense RNA. They have been successfully used to down-regulate the expression of a range of individual genes involved in the development and ripening of tomato fruit (Gray et al., 1992, Plant Molecular Biology, 19: 69-87).
Methods to increase the expression of a target gene have also been developed. For example, additional genes designed to express RNA containing the complete coding region of the target gene may be incorporated into the genome of the plant to "over-express" the gene product. Various other methods to modify gene expression are known; for example, the use of alternative regulatory sequences.
The carotenoid pathway in plants produces carotenes, lutein, xanthophylls, and pigments such as lycopene. An earlier patent application (published as EP-A-505405) describes a process to modify (inhibit or promote) the synthesis of such compounds in plants using DNA constructs comprising a DNA sequence preferably encoding a phytoene synthase enzyme (which particularly modifies colour of plant parts, especially fruit).
The late steps of carotenoid biosynthesis in plants involve the formation of xanthophylls. Little is known about the enzymology of these steps. No plant xanthophyll biosynthetic enzyme has previously been cloned. In work leading to the present invention we have purified to homogeneity a xanthophyll biosynthetic enzyme from Capsicum annuum (pepper) chromoplasts, which catalyzes the conversion of the ubiquitous 5,6-epoxycarotenoids, antheraxanthin and violaxanthin, into capsanthin and capsorubin, respectively. Due to its bifunctionality, this new enzyme has been named capsanthin-capsorubin synthase (CCS).
Both capsanthin and capsorubin are red and give colour to plant tissue. Currently both of these xanthophylls (extracted from paprika) are used as food colourants. Capsanthin and capsorubin are unique to Capsicum fruits and CCS may only be naturally expressed in species of this genus (including peppers, chillies and paprika). However, the immediate precursors for capsanthin and capsorubin (violaxanthin and antheraxanthin) are present in all green tissues.